Lee M S, Ogg S, Xu M, Parker L L, Donoghue D J, Maller J L, Piwnica-Worms H
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Mol Biol Cell. 1992 Jan;3(1):73-84. doi: 10.1091/mbc.3.1.73.
To determine how the human cdc25 gene product acts to regulate p34cdc2 at the G2 to M transition, we have overproduced the full-length protein (cdc25Hs) as well as several deletion mutants in bacteria as glutathione-S-transferase fusion proteins. The wild-type cdc25Hs gene product was synthesized as an 80-kDa fusion protein (p80GST-cdc25) and was judged to be functional by several criteria: recombinant p80GST-cdc25 induced meiotic maturation of Xenopus oocytes in the presence of cycloheximide; p80GST-cdc25 activated histone H1 kinase activity upon addition to extracts prepared from Xenopus oocytes; p80GST-cdc25 activated p34cdc2/cyclin B complexes (prematuration promoting factor) in immune complex kinase assays performed in vitro; p80GST-cdc25 stimulated the tyrosine dephosphorylation of p34cdc2/cyclin complexes isolated from Xenopus oocyte extracts as well as from overproducing insect cells; and p80GST-cdc25 hydrolyzed p-nitrophenylphosphate. In addition, deletion analysis defined a functional domain residing within the carboxy-terminus of the cdc25Hs protein. Taken together, these results suggest that the cdc25Hs protein is itself a phosphatase and that it may function directly in the tyrosine dephosphorylation and activation of p34cdc2 at the G2 to M transition.
为了确定人类cdc25基因产物在G2到M期转换过程中如何调节p34cdc2，我们在细菌中过量表达了全长蛋白（cdc25Hs）以及几种缺失突变体，它们作为谷胱甘肽-S-转移酶融合蛋白。野生型cdc25Hs基因产物被合成为一种80 kDa的融合蛋白（p80GST-cdc25），并通过以下几个标准判断其具有功能：重组p80GST-cdc25在存在放线菌酮的情况下诱导非洲爪蟾卵母细胞减数分裂成熟；将p80GST-cdc25添加到从非洲爪蟾卵母细胞制备的提取物中可激活组蛋白H1激酶活性；在体外进行的免疫复合物激酶分析中，p80GST-cdc25激活p34cdc2/细胞周期蛋白B复合物（早熟促进因子）；p80GST-cdc25刺激从非洲爪蟾卵母细胞提取物以及过量表达的昆虫细胞中分离出的p34cdc2/细胞周期蛋白复合物的酪氨酸去磷酸化；并且p80GST-cdc25水解对硝基苯磷酸。此外，缺失分析确定了一个位于cdc25Hs蛋白羧基末端的功能结构域。综上所述，这些结果表明cdc25Hs蛋白本身就是一种磷酸酶，并且它可能在G2到M期转换过程中直接参与p34cdc2的酪氨酸去磷酸化和激活。
